The overall goal of this research is to elucidate the mechanisms involved in the regulation and function of muscarinic acetylcholine receptors (mAChR) in the mammalian heart. Activation of the mAChR causes a decrease in the rate and force of contraction, and regulates the activities of adenylyl cyclase, phospholipase C, and potassium and calcium channels. The function and number of mAChR can be decreased by continued exposure of agonist. We have identified five amino acids, located in the sixth and seventh transmembrane domains and the third intracellular loop, that are essential for agonist-induced M2 mAChR internalization via a dynamic-independent receptor subtype-specific mechanism. Our hypothesis is that these crucial amino acid residues form a "sequestration-domain" which interacts with one or more intracellular proteins to target the receptor for internalization. This research will identify the protein(s) which interact with the M2 receptor and mediates its internalization. We have obtained evidence that at least one additional region of the M2 receptor which is required for morphologically detectable agonist- induced redistribution of the receptor following sequestration from the cell surface. This research will identify this region of the receptor and determine if there are intracellular proteins which interact with it. This proposal will also introduce mutations into mAChR in the heart in vivo to provide unique information on the regulation of mAChR signaling in the heart. This proposal will generate mice with targeted mutations in the M2 receptor to determine the role of the sequences identified in the previous specific aims on the regulation of mAChR expression in the heart in vivo and on the regulation of cardiovascular function. This research will provide valuable new information on the basic mechanisms regulating the expression and function of mAChR in the heart. In addition, this research may aid in understanding the etiology of a variety of cardiac abnormalities.